1. Filed of the Invention
The present invention relates to a composition for detecting or quantifying a protein in a protein-containing liquid sample (e.g., urine) at a low concentration to a trace amount.
2. Brief Description of the Background Art
It is clinically important to judge whether or not a person excessively excretes a protein, such as albumin or the like, into urine. Even in a healthy person, namely a case in which the kidney is normally functioning, an extremely small amount of a protein is excreted and the total amount thereof becomes from 50 to 100 mg per day. Additionally, it is known that the excreted amount increases after physical exercise or depending on the physical conditions, which is generally called physiological proteinuria. According to electrophoresis analyses, about 60% of the proteins excreted into urine of a healthy person is originated from the blood plasma, and albumin having a molecular weight of about 67,000 occupies about 70 to 80% thereof.
In addition to the physiological proteinuria, symptomatic proteinuria is classified into prerenal, renal and postrenal types, and the renal type is further classified into subtypes, such as glomerular origin, tubular origin, and the like. The prerenal proteinuria is a result of leakage of increased blood protein into urine caused by a functional change or disorder of specific organ tissues, and detection of such a specific component is directly connected with morbid state diagnoses. Its typical example is Bence Jones protein which is a tumor marker. Among the renal proteinuria types, the glomerular proteinuria exemplified by albumin and the like is caused by the reduced filtration function of renal glomerular basement membrane, and the tubular protein exemplified by .beta..sub.2 -microglobulin and .alpha..sub.1 -microglobulin is caused by the reduced renal tubular re-absorption ability, and both cases can be used as excellent markers for understanding the degree of renal function reduction and renal disorders. The postrenal proteinuria is a proteinuria which is expressed by, for example, bleeding, calculus, tumors, and the like, in certain organs, such as renal pelvis, urinary duct, bladder, urethra, prostate, and the like, and is used for the diagnoses of the above-described topical diseases.
Among a number of proteinuria measuring method, a protein error method using tetrabromophenol blue (TBPB) which is a pH indicator has a long history and is still established as a screening inspection method. TBPB develops yellow color in a solution of pH 2 to 3, and blue color at pH 4 or more, but becomes blue even at pH 3 when a protein is present in the solution. When a test paper, such as filter paper or the like, is impregnated with TBPB together with a buffer of pH 3 making use of this phenomenon, the tone of blue color changes depending on the existing amount of a protein, such as albumin or the like, in urine, so that the degree of proteinuria can be read out from the color tone.
However, generally, this "protein error method" cannot detect a protein of 10 to 15 mg/dl or less. Furthermore, since the method is a test paper method, the detection is out-put qualitatively, such as "-" to ".+-." to "+". Additionally, the detectable protein is generally only albumin, and globulin which occupies about 40% of plasma proteins, the above-described Bence Jones protein and the like cannot be detected.
In addition to such a qualitative measuring method, there are a number of known methods by which a protein per se can be detected quantitatively. For about 20 years, nephelometry exemplified by the Kingsbury-Clark method has been carried out as a main routine inspection method, but it has problems in that it can hardly react with proteins other than albumin similar to the case of the protein error method. Furthermore, since the method is a manual method, it takes time and labor for the measurement.
A dye binding method using a complex of a dye exemplified by pyrogallol red with a metal, such as molybdenum or the like, is now used most frequently as a quantitative determination method. According to this method, highly accurate measuring results can be obtained not only by manual handling but also by applying it to an automatic analyzer.
JP-B-4-53265 (the term "JP-B" as used herein means an "examined Japanese patent publication") discloses a colorimetric method for the determination of a trace protein, using a dye capable of forming a complex with molybdenum and of shifting its wavelength in the presence of a protein. The basic principle of this method is well known, and, when a substance which binds to molybdenum is present in a test sample, the test shows a negative value in the case of normal urine so that the absorbance becomes lower than that of the measurement of pure water.
For this point, JP-B-4-53265 takes a measure for covering molybdenum in the reagent from inhibiting substances, such as formulation of a chelating agent which binds to molybdenum or formulation of a metal ion which binds to an inhibiting substance, such as citric acid or the like, that binds to molybdenum, in advance in the reagent composition.
Also, JP-B-6-70632 discloses a colorimetric method for the determination of a trace protein, comprising using a polyhydroxybenzene sulfonphthalein dye and/or a polyhydroxybenzene phthalein dye, capable of forming a complex with tungsten and of shifting its wavelength in the presence of a protein, and a buffer for keeping the composition at an acidic pH.
Additionally, a publication "Color Reaction Between Pyrogallol Red-Molybdenum(VI) Complex and Protein", Y. Fujita, I. Mori and S. Kitano, Analytical Chemistry, 32, pp. E379-E386 (1983), describes results of screening tests of metals and dyes, which can be used as a trace protein measuring method making use of the protein error phenomenon. The dye is a polyhydroxybenzene sulfonphthalein dye or a polyhydroxybenzene phthalein dye. Examples of the metals to be screened include molybdenum(VI), bismuth(III), aluminum(III), iron(III), uranium(VI), zirconium(IV), antimony(III), tungsten(VI), cerium(III), tin(IV), zinc(II), manganese(II), mercury(II), silver(I), and cadmium(II)